NKT cells represent an innate-like lymphocyte lineage regulating disease conditions such as infection, cancer, autoimmunity and asthma through the secretion of Th1 and Th2 cytokines and chemokines. They use a semi-invariant ab TCR to recognize lipids presented by CD1d on dendritic cells, but these ligands have long remained elusive. This project investigates the nature of lipid antigens, their mode of recognition and their function in vivo. In the past funding period, we identified two conserved but structurally different NKT ligands involved in different types of anti-microbial responses. The self glycosphingolipid (GSL) iGb3 directs the developmental expansion of NKT cells and their responses against LPS-expressing bacteria, whereas aglycuronylceramides trigger responses against Gram-negative LPS-negative bacteria that would otherwise largely escape TLR detection. We will now study how immune responses triggered by these NKT ligands are integrated in the context of other microbial signals and we will use synthetic variants of these ligands to understand the basis of their cellular and subcellular targeting for antigen presentation. The specific aims are 1) Endogenous NKT ligands: their recognition and regulation in the context of autoreactive responses, particularly infectious conditions 2) Microbial NKT ligands: their recognition and interplay with Toll like receptor responses and 3) Cell biology of synthetic NKT ligands: new fluorescent lipids will be developed to address elusive questions about lipid uptake and trafficking in different cell types for antigen presentation. The project is part of a larger multidisciplinary Program Project exploring chemical, structural and functional aspects of lipid antigens during immune responses The public health implications of these studies reside in the importance of NKT cells in regulating world wide diseases such as infections by Rickettsiales, asthma, cancer and autoimmunity and in the prospects of harnessing the biological properties of their ligands for the development of new vaccine adjubants.